Project Summary Studies using heterochronic parabiosis, a surgical intervention that establishes bi-directional cross-circulation between young and old animals, strongly indicate the existence of blood-borne factors that regulate the regeneration and homeostasis of skeletal muscle, and other tissues, in an age-dependent manner. Work from my lab and others implicate the circulating proteins Growth Differentiation Factor 11 (GDF11) and Myostatin (MSTN) as candidate geronic factors responsible for the transposition of aging phenotypes in muscle in heterochronically joined mice. GDF11 and MSTN are closely related ligands of the TGF? superfamily that share 89% sequence identity in their receptor-binding domains and exhibit highly overlapping patterns of receptor binding. Yet despite these similarities, published reports suggest that these two proteins may exert opposing influences on aging muscle. In particular, we showed that systemic supplementation of GDF11 levels in aged mice (by injection of recombinant GDF11 protein) can elicit a striking reversal of age-related deficits in muscle repair capacity, remodel myofiber ultrastructure, and enhance muscle strength and endurance. GDF11 expression also has been positively correlated with lifespan in studies of multiple species. Conversely, analyses of MSTN-deficient animals suggest that MSTN acts normally to limit muscle growth and slow regeneration after injury, and that mild suppression of MSTN in mice may have a positive impact on lifespan. Importantly, these effects of GDF11 and MSTN appear to be both dose- and context-specific, which has raised some confusion and controversy regarding their respective influences on aging and health span. In this proposal, we aim to overcome prior experimental challenges to generate a clear molecular genetic understanding of the respective roles of GDF11 and MSTN in muscle aging, their mechanistic relationship to one another, and their relevance as therapeutically actionable geronic proteins. Our studies will use highly specific LC-MS/MS assays and mouse genetic models to track circulating protein levels and manipulate GDF11 and MSTN expression in young, middle-aged and aged mice, and in the young or aged partners of heterochronic parabiosis, both in steady state and in response to muscle injury. The importance of this work is underscored by human studies linking differences in serum levels of GDF11 and MSTN with clinical outcomes in several aging-related diseases. As such, our proposed work has a high potential impact for the identification and future development of promising targets to combat age-related muscle dysfunction and is directly responsive to RFA-AG-17-002: Characterization of Circulating Pro- and Anti-Geronic Proteins and Peptides.